Opioid analgesics, such as morphine and hydrocodone, are widely used for the treatment of moderate to severe pain but are also widely misused and abused. Opioid-related overdoses account for almost half of all drug overdose deaths in the United States and cause more preventable deaths every year than car crashes. Fentanyl, a highly potent mu opioid receptor (MOR) agonist, and its analogues (fentalogs) are increasingly found in drug samples from search and seizure by law enforcement and are thought to contribute to the drastic increase in opioid-induced overdose deaths since 2016. In fact, the number of deaths and drug seizures involving fentalogs is likely an underestimate, as the rapid rate at which new fentalogs are synthesized makes it difficult for law enforcement and forensic specialists to keep up. As many of these compounds are novel analogs, very little is known about their basic pharmacology in terms of receptor affinity or activity at the opioid receptors. The objective of this proposal is to evaluate novel fentalogs, many of which have been identified in illicit samples, in terms of binding affinity and receptor function at the three classical opioid receptors: MOR, the delta opioid receptor (DOR), and the kappa opioid receptor (KOR). This will be accomplished using standard competitive radioactive ligand binding and G protein turnover assays. The ability of a standard rescue therapy, naloxone (trade name Evzio or Narcan), to reverse the agonist activity of the most potent and efficacious fentalogs will also be examined; real time cAMP accumulation will be used to determine agonist function and reversal by antagonists, such as naloxone. The overarching hypothesis is that many of these novel fentalogs are more potent and efficacious than traditional opioid agonists and that standard treatments for opioid overdose may be insufficient when fentalogs are involved. Overall, this project will characterize the in vitro pharmacological properties of novel fentalogs found in street drugs and has the potential to inform best practices for preventing opioid overdose and related deaths.